| Part I Effect of Muramyl Dipeptide on the Culture and Proliferation of Dendritic Cells Derived from Bone Marrow in Children with Acute Leukemia in VitroObjective To investigate the effect of Muramyl Dipeptide (MDP) on the culture and proliferation of dendritic cells (DC) derived from bone marrow in children with acute leukemia in vitro.Methods The mononuclear cells were isolated from bone marrow in children with acute leukemia by the method of Ficoll-Hypaque and cultured with 10% fetal calf serum (FCS) (volume fraction) of RPMI-1640 in the control group, while the test groups were divided into three subgroups, cultured with MDP alone (Group 1), cytokines (Group 2), and cytokines+MDP (Group 3) respectively. The growth of cells was observed under an invert microscope everyday. We counted the numbers of different groups and detected the immunephenotypes of cells by flow cytometry in the 8th day.Results①In the 8th day, the number of cells of control group was (0.85±0.23)×105/L, the numbers of the test groups were (2.31±0.24)×105/L, (3.26±0.37)×105/L and (4.16±0.34)×105/L, all of them were higher than the control group (p<0.01). The group 1 was lower than the group 2 and group 3 (p<0.01), and the group 3 was higher than the group 2 (p<0.01).②The rates of HLA-DR+of the control group,group 1,group 2 and group 3 were (19.98±3.74)%, (37.24±4.32)%, (58.81±2.08)% and (77.48±5.57)%, the group 1 was higher than the control group (p<0.01), but lower than the group 2 and group 3 (p<0.01), and the group 3 was higher than the group 2 (p<0.01); The rates of CDla+ of the control group,group 1,group 2 and group 3 were (11.46±2.43)%, (28.71±6.64)%, (46.92±4.78)% and (57.03±3.07)%, the group 1 was higher than the control group (p<0.01), but lower than the group 2 and group 3 (p<0.01), and the group 3 was higher than the group 2 (p<0.01); The rates of CD83+ of the control group,group,1,group 2 and group 3 were (13.05±5.70)%, (36.32±5.61)%, (54.95±7.83)% and (75.70±6.67)%, the group 1 was higher than the control group (p<0.01), but lower than the group 2 and group 3 (p<0.01), and the group 3 was higher than the group 2 (p<0.01).Conclusion 1. The different groups all attained a certain amount of DCs, the number of the cells of the MDP group was lower than that of the cytokines group, and the number of the group cultured with combined application of MDP and cytokines was higher than that of the MDP group and cytokines group. It can indicate that MDP promotes the proliferation of DCs derived from bone marrow in children with acute leukemia, and the above effect is more obvious when combined with cytokines.2. The positive rates of HLA-DR+, CDla+ and CD83+ of the MDP group were higher than those of the control group, but lower than those of the cytokines group; the positive rates of the group cultured with combined application of MDP and cytokines were higher than those of the MDP group and cytokines group. It can indicate that MDP promotes the maturity of DCs derived from bone marrow in children with acute leukemia, and the above effect is more obvious when combined with cytokines. PartⅡEffect of Muramyl Dipeptide on the Immunifaction of Dendritic Cells Derived from Bone Marrow in Children with Acute LeukemiaObjective To investigate the effect of Muramyl Dipeptide (MDP) on the immunifaction of dendritic cells (DC) derived from bone marrow in children with acute leukemia.Methods The mononuclear cells were isolated from bone marrow in children with acute leukemia by the method of Ficoll-Hypaque and cultured with cytokines in the control group. The test groups contained five subgroups:used MDP 102ug/l alone (Group 1),cytokines combined with MDP of different concentration, which was put in 3rd day (Group 2,10ug/l; Group 3,102ug/l; Group 4,103ug/l; Group 5,104ug/l). The growth of cells was observed under an invert microscope everyday and collected in the 9th day. The DCs of different groups were mixed with T cells, which were separated from bone marrow in allogeneic acute lymphoblastic leukemia. Cultured with 10% FCS of RPMI-1640+rhIL-2,96 hours later, the numbers of T cells in different groups were counted and the contents of IL-12,interferon-y (IFN-y) were tested by enzyme-linked immunosorbent assay (ELISA). The DCs of different groups and T cells (cultured with 10% FCS of RPMI-1640+rhIL-2 for 6 days) and HL-60 cells were cultured for 72 hours, cytotoxicity assay was measured by methyl thiazolyl tetrazolium (MTT) method.Results (1)Mixed lymphocyte reaction①The stimulation index of T cells:When the number of stimulation cells was 1×103, the proliferation of allogeneic lymphocytes could be strongly stimulated. There was no difference among the control group, group 1 and group 2; the group3,group 4 and group 5 were higher than the control group and group 1 (p<0.01). With the increasing of the number of stimulation cells from 1×103 to 1×104, the stimulation index of T cells was rising, and the group3,group 4 and group 5 were significantly higher than the control group and group 1, but there was no difference among the control group, group 1 and group 2.②The contains of IL-12,IFN-γ:When the number of stimulation cells was 5×104, there was no difference among the contains of IL-12 of the control group, group 1 and group 2; the group3,group 4 and group 5 were higher than the control group and group 1 (P<0.01). There was no difference among the contains of IFN-γof the control group, group 1 and group 2; the group3,group 4 and group 5 were higher than the control group and group 1 (p<0.01). (2) Cytotoxicity assay showed that killing activity on HL-60 cells of T cells in test groups was higher than that of the control group, and in test groups, with the increasing of the concentration of MDP from 102ug/l to 104ug/l, the killing activity on HL-60 cells was dramatically enhanced (p<0.01), among which the killing activity of the group 5 was the strongest.Conclusion 1. When the number of DCs induced with MDP was 1×103, the proliferation of allogeneic lymphocytes could be strongly stimulated. With the increasing of the number of DCs from 1×103 to 1×104, the power of the proliferation of T cells became stronger and stronger; there was no difference between the MDP group and cytokines group. When the concentration of MDP was≥102ug/l, the above effect was obvious when combined with cytokines. It can indicate that the proliferation of T cells can be promoted when the number of DCs induced with MDP is≥1×103, and its function has MDP concentration dependence.2. The DCs induced with MDP could promote T cells to secrete IL-12 and IFN-γ, and there was no difference with the cytokines group. When the concentration of MDP was≥102ug/l, the above effect was obvious when combined with cytokines. It can indicate that DCs induced with MDP can promote T cells secretion, and has no difference between cytokines, but the combined application effect is more obvious.3. The different groups all had the killing activity on HL-60 cells, and with the increasing of the concentration of MDP from 102ug/l to 104ug/l, the killing tumor activity was dramatically enhanced. So, it can indicate that MDP can induce functional DCs to be generated from bone marrow in children with acute leukemia, and its function has MDP concentration dependence. |
PDF Full Text Request